Fgf2-binding peptides and uses thereof

ABSTRACT

FGF2-binding peptides are here described, which have been designed starting from the N-terminal region of PTX3, in particular spanning the PTX3(82-110) region. Synthetic peptides related to this sequence are able to bind FGF2 and to inhibit its pro-angiogenic activity in vitro and in vivo with no anticipated impact on innate immunity.

FIELD OF THE INVENTION

The present invention relates to Fibroblast Growth Factor-2(FGF2)-binding peptides, able to bind FGF2 and to inhibit itspro-angiogenic activity in vitro and in vivo with no anticipated impacton innate immunity.

BACKGROUND OF THE INVENTION

Pentraxins are a superfamily of proteins characterized by a pentamericstructure. The classical short-pentraxins C-reactive protein (CRP) andserum amyloid P component (SAP) are acute phase proteins in man andmouse, respectively, produced in liver in response to inflammatorymediators^(2,3). Pentraxins bind various ligands and are involved in theinnate resistance to microbes and scavenging of cellular debris andextracellular matrix components^(1,4-6).

Long-pentraxins are characterized by an unrelated N-terminal domaincoupled to a pentraxin-like C-terminal domain⁷. The prototypiclong-pentraxin PTX3^(8,9) is a 45 kD glycosylated protein predominantlyassembled in 10-20 mer multimers¹⁰. PTX3 is locally produced andreleased by different cell types, in particular by mononuclearphagocytes, dendritic cells and endothelial cells, in response toprimary inflammatory signals¹¹. Studies in ptx3^(−/−) mice have shownthat this molecule plays complex non-redundant functions in vivo,ranging from the assembly of a hyaluronic acid-rich extracellularmatrix, to female fertility and to innate immunity against diversemicroorganisms^(12,13). This is related, at least in part, to thecapacity of PTX3 to bind with high affinity the complement componentC1q, the extracellular matrix protein TSG6 and selected microorganisms,activating complement activation and facilitating pathogen recognitionby macrophages and dendritic cells^(1,14). Thus, PTX3 is a solublepattern recognition receptor with unique non-redundant functions invarious pathophysiological conditions^(1,14).

Fibroblast growth factor-2 (FGF2) is a heparin-binding growth factorthat induces cell proliferation, chemotaxis, and protease production incultured endothelial cells by interacting with high affinitytyrosine-kinase receptors (FGFRs)¹⁵. FGF2 induces angiogenesis in vivoand modulates neovascularization during wound healing, inflammation,atherosclerosis, and tumor growth¹⁶. Several molecules sequester FGF2 inthe extracellular environment, thus preventing its interaction withendothelial cell FGFRs and inhibiting its angiogenic activity (reviewedin¹⁶). Many of these inhibitors are produced/released locally and/orsystemically, thus underlying the complex tuning of the angiogenesisprocess.

Long PTX3 binds FGF2 with high affinity and specificity. Accordingly,long PTX3 inhibits FGF2-dependent endothelial cell proliferation invitro and angiogenesis in vivo¹⁷. Also, whole PTX3 inhibitsFGF2-dependent smooth muscle cell activation and intimal thickeningafter arterial injury¹⁸. Thus, PTX3 may potentially contribute to themodulation of FGF2 activity in different pathological settingscharacterized by the co-expression of the two proteins, includinginflammation, wound healing, atherosclerosis, and neoplasia. However notherapeutic use of the protein is disclosed given the unfeasibility toutilize such large molecule and to other activities of the protein. As amatter of fact, PTX3 binds C1q via the C-terminal pentraxin domain¹⁰.

At present, no biological functions have been ascribed to the PTX3N-terminus. On this basis, the authors have investigated the ability ofPTX3 N-terminus to interact with FGF2.

DESCRIPTION OF THE INVENTION

It has been found that retroviral transduced endothelial cellsover-expressing the PTX3N-terminal fragment (1-178) show reducedmitogenic activity in response to FGF2. Purified recombinant PTX3(1-178)binds FGF2 and prevents PTX3/FGF2 interaction. Also, the monoclonalantibody mAb-MNB4, that recognizes the PTX3(87-99) epitope, preventsFGF2/PTX3 interaction and abolishes the FGF2 antagonist activity ofPTX3. Surprisingly the authors found that very short peptides retainsuch activity and be useful as therapeutic drugs. Consistently,synthetic peptides PTX3(82-110), PTX3(97-110), PTX3(97-107) and PTX3(100-104) bind FGF2 and inhibit the interaction of FGF2 with whole longPTX3 immobilized to a BIAcore sensorchip, FGF2-dependent endothelialcell proliferation and angiogenesis in vivo. Thus, the data allow toidentify a very short FGF2-binding domain in the N-terminal extension ofPTX3 spanning the PTX3(97-110) region. Synthetic peptides related tothis sequence are able to bind FGF2 and to inhibit its pro-angiogenicactivity in vitro and in vivo with no anticipated impact on innateimmunity.

The main object of the present invention is therefore a FGF2-bindingpeptide of formula I:

R₁-Ala-X₁-Pro-X₂-Ala-R₂  (I)

wherein:X₁ is an amino acid selected between Arg and Lys;X₂ is an amino acid selected between Cys and Thr;R₁ is either absent or consists of the amino acid sequence selected fromSEQ ID NO: 1 and SEQ ID NO: 3;R₂ is either absent or consists of the amino acid sequence selected fromSEQ ID NO: 2 and SEQ ID NO: 4, with the following provisions:when R₁ is absent, also R₂ is absent; when R₁ is the amino acid sequenceof SEQ ID NO: 1, R₂ is the amino acid sequence of SEQ ID NO: 2; when R₁is the amino acid sequence of SEQ ID NO: 3, R₂ is an amino acid sequenceselected between SEQ ID NO: 2 and SEQ ID NO: 4; a functional derivative,a precursor or a pharmaceutically acceptable salt thereof.

Preferably, X₁ is Arg. More preferably X₂ is Cys. Eben more preferablythe peptide consists of the amino acid sequence selected among SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 10.

A further object of the invention is a conjugated chimeric peptidecomprising the peptide of formula I or functional derivatives thereof.

The term “peptide” is ordinarily applied to a polypeptidic chaincontaining from 4 to 100 or more contiguous amino acids, usually from 5to 20 contiguous amino acids. The term “functional” defines a peptideshowing FGF2-binding properties being able to greatly diminish thebiological activity of FGF2. The biological activity of FGF2 includesmitogenic and angiogenic effects. In particular, the peptides of theinvention are able to inhibit the FGF2-induced proliferation ofendothelial cells or smooth muscle cells.

The “precursors” are compounds which can be converted into the compoundsof present invention by metabolic and enzymatic processing prior orafter the administration to the cells or to the body.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of the peptides, polypeptides, oranalogs thereof, of the present invention. Salts of a carboxyl group maybe formed by means known in the art and include inorganic salts, forexample, sodium, calcium, ammonium, ferric or zinc salts, and the like,and salts with organic bases as those formed, for example, with amines,such as triethanolamine, arginine or lysine, piperidine, procaine andthe like. Acid addition salts include, for example, salts with mineralacids such as, for example, hydrochloric acid or sulfuric acid, andsalts with organic acids such as, for example, acetic acid or oxalicacid. Any of such salts should have substantially similar activity tothe peptides and polypeptides of the invention or their analogs.

The term “derivatives” as herein used refers to derivatives which can beprepared from the functional groups present on the lateral chains of theamino acid moieties or on the N-/or C-terminal groups according to knownmethods. Such derivatives include for example esters or aliphatic amidesof the carboxyl-groups and N-acyl derivatives of free amino groups orO-acyl derivatives of free hydroxyl-groups and are formed withacyl-groups as for example alcanoyl- or aroyl-groups. The presentinvention includes also peptidomimetics of the peptides alreadydisclosed, in which the nature of peptides has been chemically modifiedat the level of amino acid side chains, amino acid chirality, and/orpeptide backbone. These alterations are intended to provide FGF2-bindingagents having similar (if not improved) therapeutic, diagnostic and/orpharmacokinetic properties.

For example, when the peptide is prone to cleavage by peptidasesfollowing injection into the subject, replacement of a particularlysensitive peptide bond with a non-cleavable peptide mimetic can providea peptide more stable and thus more functional as a therapeutic.Similarly, the replacement of an L-amino acid residue is a standard wayof rendering the peptide less sensitive to proteolysis, and finally moresimilar to organic-compounds other than peptides. Also useful areamino-terminal blocking groups such as t-butyloxycarbonyl, acetyl,succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl,benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl,methoxyadipyl, methoxysuberyl, and 2,4,-dinitrophenyl. Many othermodifications providing increased efficacy, prolonged activity, easinessof purification, and/or increased half-life are known in the art.

The properties of the peptides of the invention can be maintained, oreven potentiated, in mutant peptides. Mutant peptides include amino acidsequences wherein one or more amino acid residues have beenconservatively substituted, provided they display the same biologicalactivity characterizing the present invention at equivalent or evenhigher levels, as determined by means known in the art or disclosed inthe Examples below.

In accordance with the present invention, preferred changes in themutant peptides are commonly known as “conservative” or “safe”substitutions. Conservative amino acid substitutions are those withamino acids having sufficiently similar chemical properties, in order topreserve the structure and the biological function of the molecule. Theliterature provides many models on which the selection of conservativeamino acids substitutions can be performed on the basis of statisticaland physico-chemical studies on the sequence and/or the structure ofnatural protein.

Mutant peptides may result from conventional site-directed mutagenesistechnique of the encoding DNA, from combinatorial technologies at thelevel of encoding DNA sequence (such as DNA shuffling, phagedisplay/selection) or of amino acids, from computer-aided designstudies, or any other known technique suitable thereof, which afford afinite set of substantially corresponding mutated peptides which can beroutinely obtained and tested by one of ordinary skill in the art usingthe teachings presented in the prior art and in the Examples of thepresent patent application.

Another object of the invention is a fused chimeric peptide comprisingthe peptide of formula (I) or functional derivatives thereof. TheFGF2-binding peptides being fusion and/or chimeric peptides comprise theamino acid sequence of the peptide of Formula (I) or any of theirmutants/derivatives as defined above, and an amino acid sequencebelonging to a protein sequence other than PTX3, providing additionalproperties without considerably impairing FGF2-binding activity.

Additional protein sequences which can be comprised in fusion and/orchimeric proteins can be chosen amongst membrane-bound sequences,extracellular regions of membrane-bound proteins, immunoglobulinconstant regions, multimerization domains, extracellular proteins,signal peptide-containing proteins, export signal-containing proteins.

The additional properties displayed by the fusion and/or chimericpolypeptides or peptides are an easier purification capacity, a longerlasting half-life in body fluids, or extracellular localization. Thislatter feature is of particular importance for defining a specific groupof fusion or chimeric proteins included in the above definition since itallows the peptides of the invention to be localized in the space wherenot only the isolation and purification of these peptides isfacilitated, but also where PTX3 and FGF2 naturally interact.

The choice of one or more of the sequences to be fused to theFGF2-binding peptide depends on specific use of said peptide.

As a general procedure, fusion proteins can be produced by generatingnucleic acid segments encoding them, using common genetic engineeringtechniques, and cloning in replicable vector of viral or plasmid originwhich are used to modify a Prokaryotic or Eukaryotic host cell, usingepisomal or non-/homologously integrated vectors, as well astransformation-, infection-, or transfection-based technologies. Thesevectors should allow the expression of the fusion protein including theFGF2-binding agent in the prokaryotic or eukaryotic host cell under thecontrol of their own transcriptional initiation/termination regulatorysequences, which are chosen to be constitutively active or inducible insaid cell. A cell line can be then isolated to provide a stable cellline. In particular, whenever cells modified to express the FGF2-bindingagents of the invention are directly used or administered, preferredcells are human cells, normally expressing PTX3. When the additionalprotein sequence, as in the case of the sequence of extracellular,export signal, or signal-peptide containing proteins, allows theFGF2-binding domain to be secreted in the extracellular space, the agentcan be more easily collected and purified from cultured cells in view offurther processing or, alternatively, the cells can be directly used oradministered.

When the additional protein, as in the case of the sequence ofmembrane-bound proteins, allows the immobilization of the FGF2-bindingagent on the surface of the cell, the agent can be less easily collectedand purified from the cultured cells in view of further processing butthe cells can be directly used or administered providing the agent in aform corresponding to the one of natural PTX3, possibly improving itsproperties.

The FGF2-binding peptides of the invention can be identified also bymethods of computer-aided drug design which make use of the structureand/or sequence of the peptides of the invention, or the correspondingactive mutants as defined above. The peptides of the invention may beused to study the interaction between PTX3 and FGF2 with greaterefficacy using computational modelling technologies. Suchcomputer-assisted analysis can be exploited to develop improved peptideor non-peptide mimetic drugs in the form of synthetic organic moleculesor peptides (for example, 4-20 amino acids long). Once that thesecompounds have been screened and found to be capable of binding FGF2,their use will then be assessed using cell or animal models.

The polypeptides of the invention can be in the form of activeconjugates or complex with a heterologous moiety, which may be selectedfrom cytotoxic agents, labels (e.g. biotin, fluorescent labels), drugsor other therapeutic agents, covalently bound or not, either directly orthrough the use of coupling agents or linkers. Useful conjugates orcomplexes can be generated using molecules and methods known in the art(radioactive or fluorescent labels, biotin, cytotoxic agents, drugs orother therapeutic agents). Cytotoxic agents include chemotherapeuticagents, toxin (e.g., an enzymatically active toxin of bacterial, fungal,plant, or animal origin, or fragments thereof), or a radioactive isotope(i.e., a radioconjugate). Enzymatically active toxins and fragmentsthereof that can be used include diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated proteins. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and¹⁸⁶Re.

Useful conjugates or complexes can also be generated for improving theagents in terms of drug delivery efficacy. For this purpose, thepeptides of the invention can be in the form of active conjugates orcomplex with molecules such as polyethylene glycol and other natural orsynthetic polymers (Harris J M and Chess R B, Nat Rev Drug Discov.(2003), 2(3):214-21; Greenwald R B et al., Adv Drug Deliv Rev. (2003),55(2):217-50; Pillai 0 and Panchagnula R, Curr Opin Chem. Biol. (2001),5(4):447-51). In this regard, the present invention contemplateschemically modified peptides as disclosed herein, in which the peptideis linked with a polymer. Typically, the polymer is water soluble sothat the conjugate does not precipitate in an aqueous environment, suchas a physiological environment. The conjugates used for therapy cancomprise pharmaceutically acceptable water-soluble polymer moieties.Suitable water-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono- (Cl-C10) alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone) PEG, tresyl monomethoxy PEG, PEG propionaldehyde,bis-succinimidyl carbonate PEG, propylene glycol homopolymers, apolypropyleneoxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or othercarbohydrate-based polymers. Suitable PEG may have a molecular weightfrom about 600 to about 60,000, including, for example, 5,000, 12,000,20,000 and 25,000. A conjugate can also comprise a mixture of suchwater-soluble polymers.

Examples of conjugates comprise the peptides of the invention and apolyallyl oxide moiety attached to the N-terminus of said polypeptidemoiety. PEG is one suitable polyalkyl oxide. As an illustration, thepeptides of the present invention can be modified with PEG, a processknown as “PEGylation.” PEGylation can be carried out by any of thePEGylation reactions known in the art. For example, PEGylation can beperformed by an acylation reaction or by an alkylation reaction with areactive polyethylene glycol molecule. In an alternative approach,conjugates are formed by condensing activated PEG, in which a terminalhydroxy or amino group of PEG has been replaced by an activated linker.

Another object of the invention is represented by a nucleic acidsencoding the FGF2-binding peptides of the invention, nucleic acidshybridizing with the above nucleic acids, nucleic acids havingdegenerated sequences.

The invention also includes expression vectors of viral or plasmidorigin which allows the expression of the nucleic acid of the inventionand prokaryotic or eukaryotic host cells transformed with such vectorsand stable cell lines derived therefrom, expressing the FGF2-bindingagent, which can be secreted or expressed on the membrane surface.Examples are human B cells.

FGF2-binding peptides of the invention can be produced by method whereinthe host cells above described, are cultured in an appropriate culturemedia and the FGF2-binding agent is collected.

The DNA sequence coding for the peptides of the invention can beinserted and ligated into a suitable vector. Once formed, the expressionvector is introduced into a suitable host cell, which then expresses thepeptide.

Expression of any of the recombinant peptides of the invention asmentioned herein can be effected in eukaryotic cells (e.g. yeasts,insect or mammalian cells) or prokaryotic cells, using the appropriateexpression vectors. Any method known in the art can be employed.

In order to be capable of expressing the desired protein, an expressionvector should also comprise specific nucleotide sequences containingtranscriptional and translational regulatory information linked to theDNA coding the desired protein in such a way as to permit geneexpression and production of the protein. First in order for the gene tobe transcribed, it must be preceded by a promoter recognizable by RNApolymerase, to which the polymerase binds and thus initiates thetranscription process.

There are a variety of such promoters in use, which work with differentefficiencies (strong and weak promoters).

For eukaryotic hosts, different transcriptional and translationalregulatory sequences may be employed, depending on the nature of thehost. They may be derived form viral sources, such as adenovirus, bovinepapilloma virus, Simian virus or the like, where the regulatory signalsare associated with a particular gene which has a high level ofexpression. Examples are the TK promoter of the Herpes virus, the SV40early promoter, the yeast gal4 gene promoter, etc. Transcriptionalinitiation regulatory signals may be selected which allow for repressionand activation, so that expression of the genes can be modulated.

The DNA molecule comprising the nucleotide sequence coding for theprotein of the invention is inserted into vector (s), having theoperably linked transcriptional and translational regulatory signals,which is capable of integrating the desired gene sequences into the hostcell.

The cells that have been stably transformed by the introduced DNA can beselected by also introducing one or more markers allowing for selectionof host cells containing the expression vector. The marker may alsoprovide for phototrophy to an auxotropic host, biocide resistance, e.g.antibiotics, or heavy metals such as copper, or the like. The selectablemarker gene can either be directly linked to the DNA gene sequences tobe expressed, or introduced into the same cell by co-transfection.

Additional elements of the vectors may also be useful for obtaining anoptimal production of proteins of the invention, in particular forselecting a particular cell containing plasmid or viral vector: the easewith which recipient cells, that contain the vector may be recognizedand selected from those recipient cells which do not contain the vector;the number of copies of the vector which are desired in a particularhost; and whether it is desirable to be able to “shuttle” the vectorbetween host cells of different species.

Once the vector(s) or DNA sequence containing the construct(s) has beenprepared for expression the DNA constructs) may be introduced into anappropriate host cell by any of a variety of suitable means:transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,etc.

Host cells may be either prokaryotic or eukaryotic. Preferred areeukaryotic hosts, e.g. mammalian cells, such as human, monkey, mouse,and Chinese Hamster Ovary (CHO) cells, because they providepost-translational modifications to protein molecules, including correctfolding or glycosylation at correct sites. Also yeast cells can carryout post-translational peptide modifications including glycosylation. Anumber of recombinant DNA strategies exist which utilize strong promotersequences and high copy number of plasmids that can be utilized forproduction of the desired proteins in yeast. Yeast recognizes leadersequences on cloned mammalian gene products and secretes peptidesbearing leader sequences (i.e., pre-peptides).

After the introduction of the vector(s), the host cells are grown in aselective medium, which selects for the growth of vector-containingcells. Expression of the cloned gene sequence (s) results in theproduction of the desired proteins.

Many reviews and books provides teachings on how to clone and producerecombinant proteins using vectors and Prokaryotic or Eukaryotic hostcells, such as some titles in the series “A Practical Approach”published by Oxford University Press (“DNA Cloning 2: ExpressionSystems”, 1995; “DNA Cloning 4: Mammalian Systems”, 1996; “ProteinExpression”, 1999; “Protein Purification Techniques”, 2001).

Examples of chemical synthesis technologies, which are more indicatedfor producing the FGF2-binding agent of the invention when they are inthe form of peptide or peptide mimetics, are solid phase synthesis andliquid phase synthesis. As a solid phase synthesis, for example, theamino acid corresponding to the C-terminus of the peptide to besynthetized is bound to a support which is insoluble in organicsolvents, and by alternate repetition of reactions, one wherein aminoacids with their amino groups and side chain functional groups protectedwith appropriate protective groups are condensed one by one in orderfrom the C-terminus to the N-terminus, and one where the amino acidsbound to the resin or the protective group of the amino groups of thepeptides are released, the peptide chain is thus extended in thismanner.

Solid phase synthesis methods are largely classified by the tBoc methodand the Fmoc method, depending on the type of protective group used.Typically used protective groups include tBoc (t-butoxycarbonyl), CI-Z(2-chlorobenzyloxycarbonyl), Br-Z (2-bromobenzyloxycarbonyl), Bzl(benzyl), Fmoc (9-fluorenylmethoxycarbonyl), Mbh(4,4′-dimethoxydibenzhydryl), Mtr(4-methoxy-2,3,6-trimethylbenzenesulphonyl), Trt (trityl), Tos(tosyl), Z(benzyloxycarbonyl) and Cl₂-Bzl (2,6-dichlorobenzyl) for the aminogroups; N0₂ (nitro) and Pmc (2,2,5,7,8-pentamethylchromane-6-sulphonyl)for the guanidino groups); and tBu (t-butyl) for the hydroxyl groups).After synthesis of the desired peptide, it is subjected to thede-protection reaction and cut out from the solid support. Such peptidecutting reaction may be carried with hydrogen fluoride ortri-fluoromethane sulfonic acid for the Boc method, and with TFA for theFmoc method.

The FGF2-binding agents obtained by recombinant DNA or chemicalsynthesis technologies are finally subjected to one or more steps ofpurification. Purification can be carried out by any one of the methodsknown for this purpose, i.e. any conventional procedure involvingextraction, precipitation, chromatography, electrophoresis, or the like.For example, HPLC (High Performance Liquid Chromatography) can be used.The elution can be carried using a water-acetonitrile-based solventcommonly employed for protein purification. The invention includespurified preparations of the FGF2-binding agents of the invention.Purified preparations, as used herein, refers to the preparations whichare at least 1%, preferably at least 5%, by dry weight of the compoundsof the invention.

The compounds of the invention described above (proteins, peptides,organic compounds) can be used as a medicament. Preferably as ananti-disease brought about by an altered angiogenesis. More preferablythe altered angiogenesis is provoked by an altered activation of thegrowth factor FGF2. Even more preferably the disease is selected fromthe group consisting of arthritic disease, tumor metastasis, diabeticretinopathy, psoriasis, chronic inflammation, arteriosclerosis or tumor.Preferably the tumor is selected from the group of: sarcoma, carcinoma,carcinoid, bone tumor or neuroendocrine tumor.

The compounds of the invention described above (proteins, peptides,organic compounds) can be used as as anti-disease associated withuncontrolled FGF2-dependent proliferation of fibroblasts or smoothmuscular cells, cicatrization linked to excessive fibroblastic response,and restenosis after angioplastic.

As a matter of fact the FGF2-binding peptides of the invention, oncebound to FGF2, acts as inhibitor of FGF2. Indeed the peptides are ableto inhibit the FGF2-induced proliferation of endothelial cells or smoothmuscle cells. Therefore the therapeutic potential of such molecule isthe prophylaxis and/or treatment of diseases in which an inhibition ofFGF2 is beneficial. This latter effect can be also used for reducing thepopulation of cells that express FGF2.

The FGF2-binding peptides of the invention can be used as activeingredients in pharmaceutical compositions for the prophylaxis and/ortreatment of diseases brought about by an altered angiogenesis, in whichthe altered angiogenesis is provoked by an altered activation of FGF2.Example of said diseases are: arthritic disease, tumor metastasis,diabetic retinopathy, psoriasis, chronic inflammation, arteriosclerosisor tumor, in which the tumor is, for example, sarcoma, carcinoma,carcinoid, bone tumor or neuroendocrine tumor.

The FGF2-binding agents of the invention can also be used as activeingredients in pharmaceutical compositions for the prophylaxis and/ortreatment of diseases associated with uncontrolled FGF2-dependentproliferation of fibroblasts or smooth muscular cells, such as thecicatrization linked to excessive fibroblastic response, and therestenosis after angioplastic.

The present invention also provides pharmaceutical compositioncomprising a therapeutically effective amount of the peptide of formulaI or functional derivatives thereof and suitable diluents and/orexcipients and/or adjuvants pharmaceutical for the prophylaxis and/ortreatment of the above-mentioned diseases. These pharmaceuticalcompositions can be formulated in combination with pharmaceuticallyacceptable carriers, excipients, stabilizers, or diluents. Depending onthe properties of the agent, the pharmaceutical composition can beuseful for diseases related to CD4+ T cells such as autoimmune diseases,inflammations, or infections.

Pharmaceutical compositions comprising the FGF2-binding peptides of thepresent invention include all compositions wherein said compound iscontained in therapeutically effective amount, that is, an amounteffective to achieve the medically desirable result in the treatedanimal. The pharmaceutical compositions may contain suitablepharmaceutical acceptable carriers, biologically compatible vehiclessuitable for administration to an animal (for example, physiologicalsaline) and eventually comprising auxiliaries (like excipients,stabilizers or diluents) which facilitate the processing of the activecompounds into preparations which can be used pharmaceutical.

The pharmaceutical compositions may be formulated in any acceptable wayto meet the needs of the mode of administration. The use of biomaterialsand other polymers for drug delivery, as well the different techniquesand models to validate a specific mode of administration, are disclosedin literature. Modifications of the compounds of the invention toimprove penetration of the blood-brain barrier would also be useful.

Any accepted mode of administration can be used and determined by thoseskilled in the art. For example, administration may be by variousparenteral routes such as subcutaneous, intravenous, intradermal,intramuscular, intraperitoneal, intranasal, transdermal, oral, or buccalroutes. Parenteral administration can be by bolus injection or bygradual perfusion over time. Preparations for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, andemulsions, which may contain auxiliary agents or excipients known in theart, and can be prepared according to routine methods. In addition,suspension of the active compounds as appropriate oily injectionsuspensions may be administered. Suitable lipophilic solvents orvehicles include fatty oils, for example, sesame oil, or synthetic fattyacid esters, for example, sesame oil, or synthetic fatty acid esters,for example, ethyloleate or triglycerides.

Aqueous injection suspensions that may contain substances increasing theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers. Pharmaceutical compositions include suitablesolutions for administration by injection, and contain from about 0.01to 99 percent, preferably from about 20 to 75 percent of active compoundtogether with the excipient. Compositions which can be administeredrectally include suppositories.

It is understood that the dosage administered will be dependent upon theage, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired. The dosage will be tailored to the individual subject, as isunderstood and determinable by one of skill in the art. The total doserequired for each treatment may be administered by multiple doses or ina single dose. The pharmaceutical composition of the present inventionmay be administered alone or in conjunction with other therapeuticsdirected to the condition, or directed to other symptoms of thecondition. Usually a daily dosage of active ingredient is comprisedbetween 0.01 to 100 milligrams per kilogram of body weight.

The compounds of the present invention may be administered to thepatient intravenously in a pharmaceutical acceptable carrier such asphysiological saline. Standard methods for intracellular delivery ofpeptides can be used, e.g. delivery via liposomes. Such methods are wellknown to those of ordinary skill in the art. The formulations of thisinvention are useful for parenteral administration, such as intravenous,subcutaneous, intramuscular, and intraperitoneal.

As well known in the medical arts, dosages for any one patient dependsupon many factors, including the patient's size, body surface area, age,the particular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently.

All references cited herein are entirely incorporated by referenceherein, including all data, tables, figures, and text presented in thecited references. Additionally, the entire contents of the referencescited within the references cited herein are also entirely incorporatedby reference. Reference to known method steps, conventional methodsteps, known methods or conventional methods is not in any way anadmission that any aspect, description or embodiment of the presentinvention is disclosed, taught or suggested in the relevant art.

Once understood the features of the methods and products disclosed inpresent application, the necessity and kind of additional steps can beeasily deduced by reviewing prior art, as well as the non-limitingfollowing figures and examples describing the basic details and someapplications of the invention

DESCRIPTION OF THE DRAWINGS

FIG. 1. Inhibition of FGF2 mitogenic activity by retrovirus transducedN_(term)-PTX3. (A) Western blot analysis of the conditioned medium ofmurine aortic endothelial (MAE) cells infected with EGFP, human fulllength PTX3, sC_(term)-PTX3, or N_(term)-PTX3 retroviruses. The twoimmunoreactive bands present in the sC_(term)-PTX3 lane correspond tothe glycosylated and non-glycosilated form of the recombinant protein¹⁰.(B) Retrovirus infected MAE cells were stimulated with FGF2 (0.55 nM).After 48 h, cells were trypsinized and counted. Data are expressed aspercentage of the proliferation observed in mock-infected FGF2-treatedcells (0.8 cell population doublings). (C) GM7373 cells were incubatedwith the conditioned medium from infected MAE cells and immediatelytreated with 0.55 nM FGF2. After 24 h, cells were trypsinized andcounted. Data are expressed as percentage of the proliferation observedin GM7373 cells incubated in fresh medium plus FGF2 (1.0 cell populationdoublings). In B and C, data are the mean±SD of 3 independentexperiments in triplicate.

FIG. 2. Inhibition of FGF2/PTX3 interaction by recombinantN_(term)-PTX3. (A) Recombinant 6×His-tagged N_(term)-PTX3 andC_(term)-PTX3 were expressed and purified from transformed E. coli cells(inset shows the silver staining of a SDS-PAGE gel loaded with thepurified proteins). Then, FGF2-coated wells were incubated with fulllength PTX3, N_(term)-PTX3 or C_(term)-PTX3 (all at 44 nM) for 30minutes at 37° C. The relative amount of protein bound to immobilizedFGF2 was immunodetected by incubation with a rabbit polyclonal anti-PTX3antibody as described in Material and Methods. (B) FGF2-coated wellswere incubated with biotinylated PTX3 (bPTX3, 22 nM) in the absence orin the presence of a 10 fold-molar excess of full length PTX3,N_(term)-PTX3 or C_(term)-PTX3. The amount of bPTX3 bound to immobilizedFGF2 was then measured and data were expressed as percentage of bindingmeasured in the absence of any competitor. All data are the mean±SD of 3independent experiments in triplicate.

FIG. 3. PTX3 epitope mapping. (A) Full length PTX3, N_(term)-PTX3, andC_(term)-PTX3 (200 ng/lane) were analyzed by Western blotting using themonoclonal antibodies mAb-MNB4 and mAb-16B5. (B) 128 overlapping 13-merpeptides spanning the entire human PTX3 sequence were arrayed oncellulose membranes by the SPOT-synthesis technique. Then, membraneswere probed with mAb-MNB4 (black bars) and mAb-16B5 (gray bars)antibodies and immunocomplexes were quantified by densitometric analysisof the membranes. The amino acid sequence of the PTX3 peptidesrecognized by the two antibodies are shown in underlined italic in thesingle letter code.

FIG. 4. mAb-MNB4 hampers FGF2/PTX3 interaction. (A) FGF2-coated wellswere incubated with 22 nM bPTX3 in the absence or in the presence offull length PTX3, mAb-MNB4, or mAb-16B5 (all at 220 nM). The amount ofbPTX3 bound to immobilized FGF2 was then measured and data wereexpressed as percentage of binding measured in the absence of anycompetitor. (B) GM7373 cells were incubated with FGF2 (0.55 nM) plusPTX3 (220 nM) in the absence or in the presence of mAb-MNB4 or mAb-16B5(both at 2.2 μM). After 24 h, cells were trypsinized and counted. Dataare expressed as percentage of the proliferation observed in GM7373cells incubated with FGF2 only. All data are the mean±SD of 3independent experiments in triplicate.

FIG. 5. Inhibition of FGF2/PTX3 interaction by synthetic PTX3 peptides.(A) Schematic representation of human PTX3 N-terminus and relatedsynthetic PTX3 peptides. (B) Wells coated with the indicated PTX3peptides (200 μg/well) were added with FGF2 (80 nM) and the amount ofFGF2 bound was evaluated. Data, expressed as percentage of the amount ofFGF2 bound to PTX3-coated wells, are the mean±SD of 3 independentexperiments in triplicate. (C) Upper panel: FGF2 (0.8 μM) was injectedover PTX3-coated or gelatin-coated BIAcore sensorchips. Lower panel:sensogram overlay showing the binding of increasing amounts of FGF2(0.1, 0.5, 0.8, and 1.1 μM) to immobilized PTX3. The response (in RU,Resonance Units) was recorded as a function of time. D) FGF2 (0.8 μM)was injected over a PTX3-coated BIAcore sensorchip in the presence ofincreasing concentrations of full length PTX3 (

) or synthetic peptides PTX3(82-110) (), scrambled PTX3(82-110) (∘), orPTX3(57-85) (□). The response was recorded at the end of injection andplotted as a function of the antagonist concentration. For each peptide,similar results were obtained in 2-3 independent experiments. (E) GM7373cells were incubated with FGF2 (0.55 nM) in the absence or in thepresence of PTX3 (220 nM) or of the indicated PTX3 peptides (all at 66μM). Data, expressed as percentage of the proliferation observed inGM7373 cells incubated with FGF2 only, are the mean±SD of 3 independentexperiments in triplicate.

FIG. 6. PTX3(97-110) peptide as a FGF2 antagonist. (A) Schematicrepresentation of PTX3(82-110) spanning peptides. (B) Wells coated withthe indicated PTX3 peptides (200 μg/well) were incubated with FGF2 (80nM) and the amount of bound FGF2 was evaluated. Data, expressed aspercentage of the amount of FGF2 bound to PTX3-coated wells, are themean±SD of 3 independent experiments in triplicate. (C) FGF2 (0.8 μM)was injected over a PTX3-coated BIAcore sensorchip in the presence ofincreasing concentrations of PTX3(82-110) (), PTX3(97-110) (∘),PTX3(82-101) (

), or PTX3(82-96) (▴). The response was recorded at the end of injectionand plotted as a function of the antagonist concentration. For eachpeptide, similar results were obtained in 2-3 independent experiments.(D) GM7373 cells were incubated with FGF2 (0.55 nM) in the absence or inthe presence of the indicated PTX3 peptides (all at 66 μM). Data,expressed as percentage of the proliferation observed in GM7373 cellsincubated with FGF2 only, are the mean±SD of 3 independent experimentsin triplicate. (E) FGF2 (0.8 μM) was injected over a PTX3-coated BIAcoresensorchip in the presence of increasing concentrations of PTX3(97-110)(), PTX3(100-110) (◯), PTX3(97-104) (▴), or PTX3(97-107) (▾),PTX3(104-113) (♦), PTX3(100-113) (▴), PTX3(100-104) (

). The response was recorded at the end of injection and plotted as afunction of the antagonist concentration. For each peptide, similarresults were obtained in 2-3 independent experiments.

FIG. 7. Anti-angiogenic activity of PTX3(82-110) peptide. Chicken embryochorioallantoic membrane (CAM) implanted at day 11 with alginate beadscontaining vehicle (a) or 16 pmoles FGF2 in the absence (b) or in thepresence (c) of 3 nmoles of PTX3(82-110) were photographed at day 14.Original magnification, ×5.

EXAMPLES Example 1 Materials and Methods Chemicals

Human recombinant FGF2 (accession number 09038) and PTX3 (accessionnumber swiss-prot P26022) were expressed in E. coli and Chinese hamsterovary cells, respectively, and purified as described 10,19. Synthetichuman PTX3(31-60), PTX3(57-85), and PTX3(107-132) peptides were providedby Primm (Milan, Italy), all the other peptides being provided byTecnogen (Piana di Monteverna, Caserta, Italy) (HPLC purity≧95%). Forall peptides, amino acid sequence is shown in Table 1 in the singleletter code and numbering stars from the methionine residue in position1 in the PTX3 leader sequence.

TABLE 1 Synthetic peptides spanning the human PTX3 N-terminus. SEQ IDPeptide Amino acid sequence NO: PTX3(31-60)DNEIDNGLHPTEDPTPCDCGQEHSEWDKLF 8 PTX3(57-85)DKLFIMLENSQMRERMLLQATDDVLRGEL 9 PTX3(82-110)RGELQRLREELGRLAESLARPCAPGAPAE 10 Scrambled EGLRGELRGSREAELLRQAARAPACPLPE11 PTX3(82-110) PTX3(107-132) APAEARLTSALDELLQATRDAGRRLA 12 PTX3(82-96)RGELQRLREELGRLA 13 PTX3(82-101) RGELQRLREELGRLAESLAR 14 PTX3(97-110)ESLARPCAPGAPAE 5 PTX3(97-104) ESLARPCA 15 PTX3(97-107) ESLARPCAPGA 6PTX3(100-104) ARPCA 7 PTX3(100-110) ARPCAPGAPAE 16 PTX3(82-99)RGELQRLREELGRLAESL 1 PTX3(105-110) PGAPAE 2 PTX3(97-99) ESL 3PTX3(105-107) PGA 4

Rat monoclonal antibodies directed against purified human PTX3 weredescribed previously^(10,20) (MNB1 cat. Number ALX-804-463, MNB4 cat.Number ALX-804-464, Alexis Biochemicals).

Cell Cultures

Fetal bovine aortic endothelial GM7373 cells²¹ were grown in Eagle's MEMcontaining 10% fetal calf serum (FCS). Human embryonic kidney(EcoPack2-293) packaging cells (Clontech, Calif., USA) were grown inDMEM (Life Technologies, Gaithersburg, Md.) containing 10% FCS. Balb/cmurine aortic endothelial 22106 cells (MAE cells) were obtained from R.Auerbach (University of Wisconsin, Madison, Wis.) and grown in DMEMadded with 10% FCS.

Retroviral Infection

The cDNAs encoding for human PTX3 and for the enhanced green fluorescentprotein (EGFP) were obtained as described¹⁷. The cDNAs encoding for theN-terminal fragment PTX3(1-178) (N_(term)-PTX3) and the C-terminalfragment PTX3(179-381) fused to the leader sequence for secretionPTX3(1-17) (sC_(term)-PTX3) were generated from pLX-PTX3¹⁷ by PCR andstandard cloning techniques. All cDNAs were cloned in the pBABEretroviral vector thus generating pBABE-PTX3, pBABE-N_(term)-PTX3,pBABE-sC_(term)-PTX3, and pBABE-EGFP that were used to transfect theEcoPack2-293 packaging cells in the presence of Lipofectamin 17.Transduced cells were selected with puromycin (1 μg/ml, Sigma) for 2weeks. Clones with a viral titer higher than 106 cfu/mL were used forfurther experimentation. Confluent cultures of MAE cells were thenincubated for 24 hours with the conditioned medium from pBABE-PTX3,pBABE-N_(term)-PTX3, PBABE-sC_(term)-PTX3, or pBABE-EGFP packaging cellsin the presence of polybrene (8 μg/ml, Sigma). Infected cell populationswere selected for 7 days with puromycin. Observation of EGFP-infectedcells by epifluorescence microscopy (Axiovert S100 microscope, ×10/0.25;Zeiss, Gottingen, Germany) showed that retroviral infection efficiencywas higher than 80%. To assess the levels of transgene proteinexpression and release by infected cells, cell cultures were grown underserum-free conditions for 2 days. Then, conditioned media werecollected, clarified by centrifugation, concentrated 10-fold usingCentricon YM-10 filters (Millipore), and 100 μl aliquots were probed byWestern blot analysis.

Cell Proliferation Assay

Cell proliferation assay on endothelial cells was performed asdescribed²². Briefly, GM7373 or MAE cells were seeded in 96-well dishesat 75,000 cells/cm² or 25,000 cells/cm², respectively. After 16 h, cellswere incubated in fresh medium containing 0.4% FCS plus FGF2 (0.55 nM)in the absence or in the presence of different antagonists. After 24 or48 h, respectively, cells were trypsinized and counted in a Burkerchamber.

E. coli Expression and Purification of Recombinant 6×His-tagged PTX3Fragments

N_(term)-PTX3 and C_(term)-PTX3 cDNAs were amplified from pLX-PTX3 byPCR with primers containing additional nucleotides

PTX3-N: (+) CACCGAGAACTCGGATGATTATGA 8; (SEQ ID 17) (−)TTAACCTGCCGGCAGCCAGCTCC; (SEQ ID 18) PTX3-C: (+) CACCTGTGAAACAGCTATTTTA;(SEQ ID 19) (−) TTATGAAACATACTGAGCTCC. (SEQ ID 20)

These cDNAs were cloned into the PENTR TOPO vector (PENTR DirectionalTOPO Cloning Kit, Invitrogen) and sequenced. By using the Gateway©technology (Invitrogen), N_(term)-PTX3 and C_(term)-PTX3 cDNAs frompENTR TOPO vector were then cloned into the pDEST17 vector allowing theinsertion of a 6×His-tag at the C-terminus of the recombinant proteins.E. coli BL21-AI cells (Invitrogen) were then transformed with the tworecombinant plasmids and grown at 37° C. in LB medium containing 100μg/mL ampicillin. Recombinant protein expression was induced byovernight incubation at 30° C. in the presence of 0.2% L-arabinose.After induction, cells were resuspended in binding buffer (20 mM sodiumphosphate, 0.5 M NaCl, 10 mM imidazole, pH 7.4) and lysed by sonication.Clarified supernatants were filtered through a 0.45 μm filter and loadedonto a 3.0 ml HiTrap Immobilized Metal Affinity Column (IMAC) (AmershamBiosciences) with Nickel for purification. The column was washed with100 mM imidazole in binding buffer and bound proteins were eluted with300 mM imidazole according to manufacturer's instructions. Fractionswere probed for the presence of the recombinant protein byimmunoblotting, and positive fractions were collected and desalted bygel filtration chromatography (Sephadex G25 column PD10, Amersham) inPBS. Purity of recombinant proteins was higher than 90%, as assessed bySDS-PAGE followed by silver staining of the gel (see FIG. 2A, inset).

Solid Phase Binding Assay

ELISA microplates were incubated for 16 hours at 4° C. with 100 μl/wellof 100 mM NaHCO₃, pH 9.6 (coating buffer) containing FGF2 (270 nM).Then, wells were overcoated for 2 hours at room temperature with 5% drymilk in coating buffer. Next, 100 μl aliquots of PBS containing fulllength PTX3, recombinant N_(term)-PTX3 or C_(term)-PTX3 (all at 44 nM)were incubated for 30 minutes at 37° C. onto the FGF2-coated wells.Then, wells were sequentially incubated for 1 hour at 37° C. with arabbit polyclonal anti-PTX3 antibody (1:2000 dilution) that recognizesboth PTX3 fragments with similar efficiency in Western blot and ELISA,an anti-rabbit biotinylated antibody (1:2000), and 100 p, ofstreptavidin-horseredish peroxidase (1:5000, Amersham) for 1 hour atroom temperature. Then, 100 μl/well of the chromogen substrate2,29-azinobis(3-ethylbenzthiazolinesulfonic acid) were added. Absorbancevalues were read at 405 nm in an automatic ELISA reader. In someexperiments, 100-μl aliquots of PBS containing biotin-labeled PTX3(bPTX3) (22 nM) were incubated for 30 minutes at 37° C. onto FGF2-coatedwells with or without competitors. Then, wells were washed, and theamount of bound bPTX3 was evaluated as described¹⁷. Alternatively,synthetic PTX3 peptides were immobilized on ELISA microplate wells (200μg/well) as described above. Then, FGF2 (80 nM) was added and FGF2 boundto immobilized peptides was assessed by 1 hour incubation at 37° C. witha rabbit polyclonal anti-FGF2 antibody (1:7000) followed byimmunocomplex detection as described above.

PTX3 Epitope Mapping

To identify the amino acid sequence of the epitopes binding tomonoclonal anti-PTX3 antibodies, 128 peptides were arrayed ontocellulose membranes by SPOT-synthesis technology²³. The peptides were13-amino acid long with a 3-amino acid frameshift. Membranes wereblocked with 2% milk in Tween-TBS (MBS) for 16 hours at 4° C. Afterwashing, the membranes were incubated for 90 minutes at 37° C. withmonoclonal antibodies mAb MNB4 or mAb 16B5 (both at 1:1000 dilution inMBS) and then incubated for 90 minutes at 37° C. with rabbit alkalinephosphatase-conjugated anti-rat IgG (1:30,000, Sigma) in MBS. Colorreaction was developed as described 23 and intensity of the signal wasevaluated by densitometric analysis of the membrane.

BIAcore Binding Assay

A BIAcore X apparatus (BIAcore Inc, Piscataway, N.J.) was used. Surfaceplasmon resonance was exploited to measure changes in refractive indexcaused by the ability of FGF2 to bind to PTX3 immobilized to a BIAcoresensorchip. To this purpose, PTX3 (2.2 μM) was allowed to react with aflow cell of a CM4 sensorchip that was previously activated with 50 μlof a mixture of 0.2 M N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride and 0.05 M N-hydroxysuccinimide. These experimentalconditions allowed the immobilization of 5,000 resonance units (RUs),corresponding to approximately 0.1 pmoles of PTX3. Similar results wereobtained for immobilization of gelatin, here used as a negative controland for blank subtraction. Increasing concentrations of FGF2 with orwithout synthetic PTX3 peptides were then injected in dilution buffer(PBS plus 0.005% surfactant P20, 5.0 μg/mL CaCl₂ and MgCl₂) over thePTX3 surface for 4 minutes (to allow their association with immobilizedPTX3) and then washed until dissociation was observed.

Chicken Embryo Chorioallantoic Membrane (CAM) Assay

Alginate beads (5 μl) containing vehicle or 16 pmoles of FGF2 with orwithout synthetic PTX3 peptides were prepared as described 24 and placedon top of the CAM of fertilized White Leghorn chicken eggs at day 11 ofincubation (10 eggs per experimental group). After 72 hours, bloodvessels converging towards the implant were counted by two observers ina double-blind fashion under a stereomicroscope (STEMI-SR, x2/0.12;Zeiss).

Results The N-terminal Region of PTX3 Binds FGF2

PTX3 protein is characterized by a C-terminal 203-amino acid domain(C_(term)-PTX3) that shares homology with the classic short-pentraxinsCRP and SAP and by an N-terminal 178-amino acid extension(N_(term)-PTX3) that does not show any significant homology with anyother known protein 8. In the attempt to identify the antiangiogenic,FGF2-binding domain(s) of PTX3, the two C_(term) or N_(term)-PTX3portions were assessed for their capacity to interact with FGF2.

Previous observations had shown that the overexpression of full lengthPTX3 results in the inhibition of FGF2-dependent proliferation inendothelial cells due to the binding of released PTX3 to the exogenousgrowth factor and its sequestration in the extracellular milieu 17. Onthis basis, murine aortic endothelial (MAE) cells were infected withretroviruses harboring human full length PTX3, the PTX3 N-terminalextension N_(term)-PTX3, or the PTX3 C-terminus fused to the PTX3 leadersequence for secretion (sC_(term)-PTX3). Control cells were infectedwith an EGFP-harboring retrovirus. Infected cells overexpressed andreleased the corresponding proteins in similar amounts (FIG. 1A) andshowed a similar rate of growth under basal conditions. However,N_(term)-PTX3 overexpression caused a significant decrease in thecapacity of infected cells to proliferate in response to exogenous FGF2,similar to full length PTX3-overexpression (FIG. 1B). No inhibition wasinstead exerted by sC_(term)-PTX³ overexpression when compared tocontrol EGFP-infected cells.

To further assess the capacity of N_(term)-PTX3 to act as aFGF2-antagonist, conditioned media of infected MAE cells were evaluatedfor the capacity to affect FGF2-dependent proliferation of endothelialGM7373 cells (FIG. 1C). As anticipated, incubation of GM7373 cells withFGF2 in the presence of the conditioned medium of N_(term)-PTX3-infectedor PTX3-infected MAE cells caused a significant inhibition of themitogenic activity of the growth factor, whereas the conditioned mediaof sC_(term)-PTX3-infected and EGFP-infected MAE cells were ineffective(FIG. 1C). None of the conditioned media caused a significant inhibitionof GM7373 cell proliferation triggered by 10% FCS, thus confirming thespecificity of the effect.

To confirm that the FGF2-antagonist activity of N_(term)-PTX3 was due toits capacity to interact directly with the growth factor, N_(term)-PTX3was expressed and purified from transformed E. coli cells as arecombinant 6×His-tagged protein; purified recombinant 6×His-taggedC_(term)-PTX3 was used as a control (FIG. 2A, inset). When assessed forFGF2 interaction, full length PTX3 and the recombinant N_(term)-PTX3fragment showed the capacity to bind FGF2 immobilized to non-tissueculture plastic. No interaction was instead observed with recombinantC_(term)-PTX3 (FIG. 2A). Accordingly, a 10 fold-molar excess ofrecombinant N_(term)-PTX3 or of full length PTX3, but not ofC_(term)-PTX3, prevented the binding of biotinylated PTX3 (bPTX3) toimmobilized FGF2 (FIG. 2B).

Taken together, these results implicate the N-terminal region of PTX3for FGF2 interaction.

Inhibition of FGF2/PTX3 Interaction by a Monoclonal Anti-N_(term)-PTX3Antibody

The screening of a set of rat monoclonal antibodies raised against humanfull length PTX3 identified the antibodies mAb-MNB4²⁰ (MNB4 cat. NumberALX-804-464, Alexis Biochemicals) and mAb-16B5¹⁰ (MNB1 cat. NumberALX-804-463, Alexis Biochemicals) that selectively bind recombinantN_(term)-PTX3 and C_(term)-PTX3, respectively, in a Western blot (FIG.3A).

To map the PTX3 epitopes recognized by the two antibodies, the authorstook advantage of the SPOT-synthesis technique²³ by which 128overlapping 13-mer peptides spanning the entire human PTX3 sequence werearrayed on a cellulose membrane. When the membrane was probed with thetwo monoclonal antibodies, immunocomplex detection revealed thatmAb-MNB4 recognizes the epitope PTX3(87-99) present in the N-terminalextension of PTX3 whereas mAb-16B5 recognizes the epitope PTX3(306-312)located in the C-terminal region of PTX3 (FIG. 3B).

When tested for the capacity to affect FGF2/PTX3 interaction, mAb-MNB4,but not mAb-16B5, prevents the capacity of bPTX3 to bind immobilizedFGF2, similar to a molar excess of free unlabeled PTX3 (FIG. 4A).Accordingly, mAb-MNB4 abolishes the capacity of full length PTX3 toinhibit the mitogenic activity exerted by FGF2 in endothelial GM7373cells whereas mAb-16B5 is ineffective (FIG. 4B). Thus, mAb-MNB4recognizing the N-terminal PTX3(87-99) epitope neutralizes FGF2/PTX3interaction.

Synthetic N_(term)-PTX3-related Peptides as FGF2 Antagonists

To further define the FGF2-binding region in the N-terminal extension ofPTX3, the authors evaluated the FGF2-antagonist activity of thesynthetic peptide PTX3(82-110), that contains the PTX3(87-99) epitoperecognized by the neutralizing mAb-MNB4 (see above), together with threedistinct synthetic peptides PTX3(31-60), PTX3(57-85), and PTX3(107-132)partially spanning the N_(term)-PTX3 amino acid sequence (FIG. 5A).

In a first set of experiments, the four synthetic PTX3 fragments wereassessed for their capacity to interact with FGF2 in a solid phasebinding assay. As shown in FIG. 5B, free FGF2 binds to PTX3(82-110)immobilized onto non-tissue culture plastic but not to immobilizedPTX3(31-60) or PTX3(57-85), showing only a limited interaction withimmobilized PTX3(107-132).

Next, surface plasmon resonance was exploited to assess the ability ofthe four peptides to affect FGF2/PTX3 interaction. Results show thatFGF2 (0.8 μM) binds to PTX3 immobilized to a BIAcore sensorchip withhigh capacity (350-400 RU bound at the end of the injection phase) (FIG.5C, upper panel). Specificity of the interaction is demonstrated by thelack of binding to a gelatin-coated sensorchip. Also, increasingconcentrations of FGF2 (from 0.1 to 1.1 μM, FIG. 5C, lower panel) wereinjected over the PTX3 surface to evaluate the kinetic parameters ofFGF2/PTX3 interaction. The binding data demonstrate that the interactionoccurs with a kinetic dissociation constant (k_(off)) of 6×10⁻⁵ s⁻¹ anda kinetic association constant (k_(on)) of 0.2×10 s⁻¹ M⁻¹, thusresulting in a K_(d) value equal to 0.3×10⁻⁶ M⁻¹. On this basis, thefour synthetic PTX3 peptides were assessed for their capacity tosequester FGF2 in the mobile phase, thus preventing its interaction withthe PTX3 sensorchip. As shown in FIG. 5D, PTX3(82-110) inhibits thebinding of FGF2 to the PTX3 surface in a dose-dependent manner with apotency 30 times lower than that shown by free full length PTX3 (ID₅₀equal to 1.0 μM and 30 μM for free PTX3 and PTX3(82-110) peptide,respectively). Under the same experimental conditions, no inhibitoryeffect was instead exerted by PTX3(31-60), PTX3(57-85), andPTX3(107-132) peptides (FIG. 5D and other collected data). Also, ascrambled synthetic peptide with amino acid composition equal toPTX3(82-110) [sPTX3(82-110), Table 1] showed a limited inhibitory effect(ID₅₀>3000 μM) (FIG. 5D), thus indicating that the primary amino acidsequence in PTX3(82-110) is of importance for FGF2 interaction.

The capacity of PTX3(82-110) peptide to bind FGF2 prompted the authorsto assess its ability to act as a FGF2-antagonist. When tested onendothelial GM7373 cells, both full length PTX3 and PTX3(82-110) inhibitthe mitogenic activity exerted by exogenous FGF2, whereas scrambledPTX3(82-110), PTX3(31-60), PTX3(57-85), and PTX3(107-132) peptides wereineffective (FIG. 5E). Dose-response curves confirmed that theFGF2-antagonist activity of PTX3(82-110) was dose-dependent (ID₅₀ equal30 μM and 30 nM for PTX3(82-110) and PTX3, respectively).

Identification of a Minimal Linear FGF2-Binding Sequence in theN-Terminal Extension of PTX3

Taken together, the above data indicate that the linear amino acidsequence 82-110 in the N-terminal extension of PTX3 plays an importantrole in FGF2 interaction. In the attempt to identify a minimal linearFGF2-binding sequence, three overlapping synthetic peptides PTX3(82-96),PTX3(82-101), and PTX3(97-110) spanning the entire PTX3(82-110) sequence(FIG. 6A and Table 1) were evaluated for the capacity to interact withFGF2 in a solid phase binding assay. Under the same experimentalconditions, free FGF2 binds to immobilized PTX3(97-110), as well as toparental PTX3(82-110) and full length PTX3, without interacting withPTX3(82-96) or PTX3(82-101) (FIG. 6B). Accordingly, PTX3(97-110) bindsFGF2 in the mobile phase, thus preventing its interaction with PTX3immobilized to a BIAcore sensorchip (FIG. 6C). The inhibitory activityof PTX3(97-110) was similar to that shown by the parental peptidePTX3(82-110), whereas PTX3(82-96) and PTX3(82-101) were ineffective(FIG. 6C). In keeping with these observations, PTX3(97-110), but notPTX3(82-96) or PTX3(82-101), inhibits the mitogenic activity exerted byFGF2 in endothelial GM7373 cells (FIG. 6D).

To further investigate the minimal linear FGF2-binding sequence spanningthe peptide PTX3(97-110), the authors analysed the binding of thefollowing shorter peptides to FGF2 by measuring its interaction withPTX3 immobilized to a BIAcore sensorchip: PTX3(97-107), PTX3(97-104),PTX3(100-104) and PTX3(100-110), (FIG. 6E). Peptides PTX3(97-107) andPTX3(100-104) showed a significant binding to FGF2. In contrast peptidesPTX3(97-104) and PTX3(100-110) did not prevent the binding of free FGF2to PTX3 immobilized to a BIAcore sensorchip. (FIG. 6E). Thus,PTX3(100-104) appears to represent the minimal linear FGF2-binding aminoacid sequence in the N-terminal extension of PTX3. Accordingly,PTX3(100-104) inhibits FGF2-induced endothelial cell proliferation.

Synthetic N_(term)-PTX3-related Peptides Inhibit the Angiogenic Activityof FGF2

To assess the capacity of N_(term)-PTX3-related peptides to affectFGF2-induced neovascularization in vivo, gelatin sponges adsorbed withFGF2 alone or added with PTX3 peptides were implanted on the top of 11day-old chick embryo CAMs. As shown in FIG. 7, alginate beads adsorbedwith FGF2 (16 pmoles/embryo) exert a potent angiogenic response whencompared to beads adsorbed with vehicle (macroscopic vessels convergingtowards the implant being equal to 44±7 and 11±5 vessels/embryo for thetwo experimental groups, respectively). In keeping with the in vitroobservations, the in vivo FGF2-dependent angiogenic response wassignificantly reduced (28±5 vessels/embryo, p<0.05 by ANOVA) by theaddition of 3.0 nmoles of PTX3(82-110) peptide in the FGF2 implants(FIG. 7). Accordingly, 80 nmoles of PTX3(97-110) caused a 50% inhibitionin the angiogenic response triggered by FGF2; no effect was insteadexerted by PTX3(82-96).

Discussion

The authors show that FGF2 interaction is mediated by the N-terminalextension on PTX3. Also, experiments performed with neutralizingmonoclonal antibodies and synthetic PTX3-related peptides identify theamino acid linear sequence PTX3(97-110) as responsible for thisinteraction. These conclusions are based on the following experimentalevidences: i) the short-pentraxins CRP and SAP are inefficient FGF2binders¹⁷ despite their sequence homology with the PTX3 C-terminus⁷; ii)retroviral transduction of the N-terminal fragment PTX3(1-178)(N_(term)-PTX3), but not of sC_(term)-PTX3, inhibits the mitogenicactivity exerted by exogenous FGF2 in endothelial cells; iii)recombinant N_(term)-PTX3, but not C_(term)-PTX3, binds to immobilizedFGF2 and inhibits PTX3/FGF2 interaction; iv) the monoclonal antibodymAb-MNB4, mapping the linear epitope PTX3(87-99), prevents FGF2/PTX3interaction and abolishes the FGF2 antagonist activity of PTX3 inendothelial cells; v) the synthetic peptide PTX3(82-110) and the shorterpeptides PTX3(97-110), PTX3(97-107) and PTX3(100-104), but not otherpeptides based on different regions of the PTX3 N-terminus, preventFGF2/PTX3 interaction by binding FGF2, thus inhibiting FGF2-dependentendothelial cell proliferation in vitro and angiogenesis in vivo.

PTX3 is produced by macrophages²⁷, fibroblasts⁹, myoblasts²⁸,microglia²⁹, and endothelial cells⁸, indicating that it may exertparacrine and autocrine functions on endothelium. Similarly, variousstimuli, including the inflammatory mediators IL-1 and nitricoxide^(30,31) induce FGF2 expression in endothelial cells that undergoan autocrine loop of stimulation. Thus, endothelial cells and other celltypes can express both PTX3 and FGF2. Thus, PTX3 produced byinflammatory cells or by endothelial cells themselves may affect theautocrine and paracrine activity exerted by FGF2 on endothelium in vitroand in vivo. This should allow a fine tuning of neovascularization viathe production of both angiogenesis inhibitors and stimulators.

FGF2 is a pleiotropic growth factor that stimulates various cell typesof endodermal and mesodermal origin³². Therefore, the role exerted byFGF2 in various pathophysiological conditions is not limited to itsangiogenic activity. For instance, FGF2 stimulates the migration andproliferation of fibroblasts during wound healing and of smooth musclecells during atherosclerosis^(33,34) and restenosis³⁵. Also, it mayfavor neuronal cell survival and glia cell proliferation in the injuredcentral nervous system³⁶. In all these conditions, the concomitantproduction of PTX3^(37,38) modulate the activity exerted by FGF2 onthese cells. Indeed, PTX3 inhibits FGF2-dependent smooth muscle cellactivation in vitro and intimal thickening after arterial injury invivo¹⁸.

In conclusion, the authors demonstrate for the first time that PTX3N-terminus is involved in FGF2 interaction. PTX3 is a multifunctionalsoluble pattern recognition receptor at the crossroads between innateimmunity, inflammation, matrix deposition, and female fertility. Itexerts its multifunctional activity by interacting with numerous ligandswith distinct molecular properties.

REFERENCES

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1. A FGF2-binding peptide of formula I:R₁-Ala-X₁-Pro-X₂-Ala-R₂  (I) wherein: X₁ is an amino acid selectedbetween Arg and Lys; X₂ is an amino acid selected between Cys and Thr;R₁ is either absent or consists of the amino acid sequence selected fromSEQ ID NO: 1 and SEQ ID NO: 3; R₂ is either absent or consists of theamino acid sequence selected from SEQ ID NO: 2 and SEQ ID NO: 4, withthe following provisions: when R₁ is absent, also R₂ is absent; when R₁is the amino acid sequence of SEQ ID NO: 1, R₂ is the amino acidsequence of SEQ ID NO: 2; when R₁ is the amino acid sequence of SEQ IDNO: 3, R₂ is an amino acid sequence selected between SEQ ID NO: 2 andSEQ ID NO: 4; a precursor or a pharmaceutically acceptable salt thereof.2. The peptide according to claim 1, wherein X₁ is Arg.
 3. The peptideaccording to claim 1, wherein X₂ is Cys.
 4. The peptide according toclaim 1, which consists of the amino acid sequence selected among SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO:
 10. 5. A fused chimericpeptide comprising the peptide according to claim
 1. 6. The fusedchimeric peptide according to claim 5 wherein the fused amino acidsequence belongs to a protein sequence other than human PTX3, theprotein selected from the group of: membrane-bound proteins,extracellular regions of membrane-bound proteins, immunoglobulinconstant regions, multimerization domains, extracellular proteins,signal peptide-containing proteins, export signal-containing proteins.7. A conjugated chimeric peptide comprising the peptide according toclaim
 1. 8. A nucleic acid molecule encoding the peptide according toclaim 1, or hybridizing with the above nucleic acid, or including adegenerated sequence thereof.
 9. An expression vector comprising thenucleic acid molecule according to claim
 8. 10. A host cell transformedwith the expression vector according to claim
 9. 11. The host cellaccording to claim 10 wherein the peptide is secreted or expressed onthe membrane surface of the cell.
 12. A medicament comprising a peptideaccording to claim
 1. 13. A method for the prophylaxis and/or treatmentof a disease brought about by an altered angiogenesis the methodcomprising administering a therapeutically effective amount of a peptideaccording to claim 1 to an individual in need thereof.
 14. The peptidemethod of claim 13 in which the altered angiogenesis is provoked by analtered activation of the growth factor FGF2.
 15. The method of claim13, in which the disease is selected from the group consisting ofarthritic disease, tumor metastasis, diabetic retinopathy, psoriasis,chronic inflammation, arteriosclerosis and tumor.
 16. The method ofclaim 15 in which the tumor is selected from the group of: sarcoma,carcinoma, carcinoid, bone tumor or neuroendocrine tumor.
 17. A methodfor the prophylaxis and/or treatment of a disease associated withuncontrolled FGF2-dependent proliferation of fibroblasts or smoothmuscular cells, cicatrization linked to excessive fibroblastic response,and restenosis after angioplastic, the method comprising: administeringa therapeutically effective amount of a peptide according to claim 1 toan individual in need thereof.
 18. A pharmaceutical compositioncomprising a therapeutically effective amount of the peptide accordingto claim 1 and suitable diluents and/or excipients and/or adjuvants.